From owner-drosophila@net.bio.net Sun Sep 10 23:00:00 1995 Path: biosci!PSU.EDU!gxt5 From: gxt5@PSU.EDU (Graham Thomas) Newsgroups: bionet.drosophila Subject: Re: (no subject) Date: 11 Sep 1995 11:24:57 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 51 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >Here's a simple question, which I hope someone has a simple answer to. >We are staining whole-mount embryos with an antibody, and then with an >HRP-conjugated second antibody. We are getting signal, but there is >some background, presumably from endogenous peroxidases. The standard >protocol to inactivate peroxidases is to incubate with 3% H2O2 in >methanol, just after devitellinizing. However, the antigen is methanol >sensitive, so we are hand-peeling the embryos. I've been told that >treatment of specimens with 3% H2O2 in aqueous solutions results in >violent bubbling and destruction of the specimen. We will be trying a >few things, but if anyone has a successful method for inactivating >endogenous peroxidase without methanol, please let me know. > >***************************************************************** >* Alan C. Christensen, Ph.D. "...there are those rare * >* School of Biological Sciences characters who study the * >* University of Nebraska unknown product of an * >* Lincoln, NE 68588-0118 unknown gene. These we * >* Phone 402-472-0681 call lunatics." * >* FAX 402-472-2083 -Sidney Brenner * >***************************************************************** Not exactly a direct solution, but something you might want to bear in mind. We stain with phalloidin for F-actin, which similarly cannot be exposed to MeOH. Through fooling around we discovered that one can devitellinize in Ethanol (same procedure) with a slight compromise in morphology, and that this does not perturb phalloidin staining. You might want to try and see a) if your antigen can withstand EtOH and then b) if H2O2 inactivation of endogenous peroxidases works in EtOH (this I have not done). FliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFlies l i PLEASE TRY THIS URL e AND TELL SOME GRADUATING SENIORS ABOUT IT s http://www.bmb.psu.edu/thomas/cdb/cdbhome.htm F l i Graham Thomas, e Departments of Biology and Biochemisty/Molecular Biology, s The Pennsylvania State University, F 208 Mueller Laboratory, l University Park, PA, 16802 i e Email GXT5@ PSU.EDU s Telephone (814)-863-0716 F Fax (814)-865-9131 l WWW http://millreef.mcb.psu.edu/thomas i esFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFliesFli .